Michele Maiello

Increased expression of basement membrane components in human endothelial cells cultured in high glucose.

Although the degree of hyperglycemia is a powerful and independent risk factor for diabetic microvascular disease, it has not been established if and how high glucose per se can induce the typical lesions of microangiopathy. We have investigated in human vascular endothelial cells the expression of messenger RNA (mRNA) for collagen type IV and fibronectin, the two glycoproteins characteristically increased in diabetic basement membranes. In 12 confluent primary cultures exposed for 11 +/- 1 d (mean +/- SD) to 30 mM glucose and exhibiting cell number and thymidine incorporation similar to control cultures, the levels of collagen IV and fibronectin mRNA were, respectively, 238 +/- 140 and 221 +/- 231 percent of control (P less than 0.01). The effects of high glucose were selective (the levels of collagen I and c-myc mRNA remained unchanged), independent of the proliferative activity of the cultures and of the plating substratum, and maintained throughout multiple passages. However, several days of exposure to high glucose were required before their appearance. These observations establish that high glucose is a perturbation sufficient to mimic the effects of diabetes on the regulation of basement membrane components and propose that modifications in gene expression may pertain to the chain of events leading to diabetic angiopathy.

Increased Expression of Tissue Plasminogen Activator and Its Inhibitor and Reduced Fibrinolytic Potential of Human Endothelial Cells Cultured in Elevated Glucose

In diabetic patients, elevated plasma levels of t-PA and PAI-1 accompany impaired fibrinolysis. To identify mechanisms for these abnormalities, we examined whether vascular endothelial cells exposed to high glucose upregulate t-PA and PAI-1 production and whether ambient PA activity is decreased concomitantly. In 17 cultures of human umbilical vein endothelial cells grown to confluency in 30 mM glucose, the t-PA antigen released to the medium in 24 h was (median) 52 ng/106 cells (range 10–384) and the PAI-1 antigen was 872 ng/106 cells (range 217–2074)—both greater (P < 0.02) than the amounts released by paired control cultures grown in 5 mM glucose—29 ng/106 cells (range 7.5–216) and 461 ng/106 cells (range 230–3215), respectively. In the presence of high glucose, the steady-state levels of t-PA and PAI-1 mRNAs were increased correspondingly (median 142 and 183% of control, respectively, P < 0.05); high glucose per se and hypertonicity contributed to the upregulation in additive fashion. The PA activity of conditioned medium from cultures exposed to high glucose was 0.4 IU/ml (range 0.2–0.6), which was significantly lower (P < 0.02) than the PA activity of control medium (0.5 IU/ml, range 0.2–0.9). No difference was observed when comparing the PA activities of acidified conditioned media, expected to be depleted of inhibitors. Thus, high glucose coordinately upregulates endothelial t-PA and PAI-1 expression through effects exerted at the pretranslational level and enhanced by even mild degrees of hypertonicity. The decrease in ambient fibrinolytic potential may reflect an overwhelming effect of the increased availability of PAI-1. These findings propose a contributory mechanism for the fibrinolytic abnormalities of diabetes and the thrombotic tendency of the hyperglycemic hyperosmolar state.

Increased expression of basement membrane collagen in human diabetic retinopathy.

Basement membrane thickening is the most prominent and characteristic feature of early diabetic microangiopathy. Unknown is not only the causative process but also whether the thickening reflects increased synthesis of specific components. Because collagen type IV is uniquely present in basement membranes and represents their predominant structural element, we studied its expression in retinas obtained postmortem from five patients with 8 +/- 3 yr of diabetes and six nondiabetic controls. The collagen IV transcript proved to be rare in adult human retina and undetectable by Northern analysis. We thus identified a set of primers and conditions to detect the transcript by the reverse transcriptase polymerase chain reaction and to measure its level relative to an endogenous internal standard (beta-actin mRNA). In the diabetic patients the levels of collagen IV mRNA were increased twofold over levels in controls, whereas the actin mRNA levels were similar in the two groups. Hence, the collagen IV/actin ratio was 0.53 +/- 0.15 in diabetic samples and 0.24 +/- 0.09 in control samples (P = 0.004). These results indicate that diabetes induces a twofold increase in the expression of collagen IV by the cells that synthesize basement membranes in the adult retina (vascular cells). Insofar as high ambient glucose in vitro elicits the same effect, it may be proposed that basement membrane thickening in diabetes results from enhanced synthesis of specialized component molecules sustained by hyperglycemia.

Basal Synthesis of Heat Shock Protein 70 Increases with Age in Rat Kidneys

The heat shock protein (HSP) system is a mechanism of cell defense induced by stress, constitutively expressed during basal conditions and essential to the maintenance of cellular integrity. Acutely induced HSP synthesis decreases with aging, but the effect of age on the basal expression of HSP70 has not been specifically addressed so far. The aim of this work is to study the age-dependent basal concentrations of HSP70 mRNA in rat kidneys. In 8 young (2–3 months), 6 adult (6–11 months) and 6 old male Wistar rats (22–27 months), steady-state concentrations of HSP70 and γ-actin mRNA and of rRNA were measured. Pentosidine was measured by HPLC. The basal, unstimulated HSP70 mRNA is increased in young and old rats compared with adult subjects [young: 182% of adult levels (100–299), old: 167% of adults (142–209); p < 0.005]. The amount of pentosidine increases with age (young: 0.6 ± 0.1; adult: 1.65 ± 0.15; old: 2.3 ± 0.3 pmol/mg of protein; p < 0.0001). It seems likely that different mechanisms are responsible for the increased HSP70 basal synthesis in both the young and old animals. The prevalence of anabolic activity can trigger the increased basal production of HSP70 in young rats. The accumulation of posttranslational modified proteins, documented by pentosidine, can chronically enhance HSP70 synthesis in aged animals. The suppression of the synthesis of other proteins accompanying HSP-selective production might contribute to the impairment of specific cell functions in aging.

Modification of Tissue-Factor mRNA and Protein Response to Thrombin and Interleukin 1 by High Glucose in Cultured Human Endothelial Cells

Because diabetic vascular disease is accompanied by a state of hypercoagulability, manifested by increased thrombin activity and foci of intravascular coagulation, we investigated whether a specific procoagulant property of the endothelium—production and surface expression of tissue factor—is modified by elevated glucose concentrations. In unperturbed human vascular endothelial cells, tissue factor mRNA and expression of the functional protein were undetectable and werenot induced by 10–12 days of exposure to 30 mM glucose. In thrombin-stimulated cultures, tissuefactor expression was related inversely to cellular density, with confluent cultures producing (per 105 cells) half the amount of tissue factor measured in sparse cultures. Cells exposed to high glucose and studied when cell number and thymidine incorporation were identical to control cells manifested increased tissue-factor mRNA level and functional protein production in response to thrombin (P = .002). This effect was not attributable to hypertonicity and was not observed after short exposure to high glucose. In contrast, the tissue-factor response to interleukin 1, a modulator of endothelial function in the context of host defense, was decreased in cells cultured in high glucose (P = .04). These findings indicate that exposure to high glucose can alter tissue-factor gene expression in perturbed vascular endothelium. The reciprocal effects of high glucose on the tissue-factor response to thrombin and interleukin 1 points to different pathwaysof tissue-factor stimulation by the two agents and suggests functional consequences pertinent to the increased thrombin activity and compromised host-defense mechanisms observed in diabetes.

I.V. Glucose tolerance test: Correlation between FFA, glucose and IRI in normal, obese and diabetic subjects

SummaryInsulin response and FFA behavior have been evaluated during an IVGTT in 63 subjects of whom 18 were normal, 31 were obese (with varying degrees of carbohydrate tolerance) and 14 were mild non insulin-dependent diabetics. The extreme reduction of insulin secretion in the early phase (Δ 0–15 min) and the less severe impairment of the late phase (Δ 15–60 min) have been confirmed; obese subjects showed on the average an active insulin response to venous loading; this was more marked and more consistent in the late phase. Compared to controls, FFA concentration both in basal conditions and during IVGTT was progressively higher in obese and diabetic patients. When analyzing the interplay between IRI, KG and FFA in the course of IVGTT, it was observed that: (1) a close correlation exists between KG and early insulin response (r=0.72); (2) a correlation between Δ IRI 0–15 min and percentage decrease of FFA at 45 min is found only in normal subjects; (3) a negative highly significant correlation is found between Kg and mean FFA plasma level 0–60 min. This last correlation is evidence of the important role played by FFA in carbohydrate tolerance. The conflicting results reported by others have been discussed.

Exploration of the early insulin response by two small successive loads of I.V. glucose in normal and obese subjects.

SummaryTwo 5 g glucose loads at 1-h interval were given to healthy controls and obese subjects with slightly altered or normal OGTT in order to explore the capacity of restoration of the ‘rapid insulin response’ to i.v. glucose. In the normal subjects, the two successive loads gave rise to identical responses as far as maximum increase (Δmax), average increase at 2–5 min (Δ2–5 min), area of increase 0–15 min (Δ0–15 min) for both glucose and IRI, were concerned. Obese subjects could be divided on the basis of their insulin response to the first load into normal responders (group I) and high-responders (group II). In group I obese subjects, the responses to the second load were identical to those to the first. In group II obese subjects Δmax, Δ2–5 min and Δ0–15 min of the insulin response to the second load were reduced as compared to the first.